1. Field of the Invention
The present application discloses a systematic and efficient method for establishing stable neural stem cell lines and neuronal progenitor lines. The resulting cell lines provide robust, simple, and reproducible cultures of human and other mammalian neurons in commercially useful mass quantities while maintaining normal karyotypes and normal neuronal phenotypes.
2. Description of the Related Art
A developing fetal brain contains all of the cells germinal to the cells of an adult brain as well as all of the programs necessary to orchestrate them toward the final network of neurons. At early stages of development, the nervous system is populated by germinal cells from which all other cells, mainly neurons, astrocytes and oligodendrocytes, derive during subsequent stages of development. Clearly such germinal cells that are precursors of the normal brain development would be ideal for all gene-based and cell-based therapies if these germinal cells could be isolated, propagated and differentiated into mature cell types.
The usefulness of the isolated primary cells for both basic research and for therapeutic application depends upon the extent to which the isolated cells resemble those in the brain. Just how many different kinds of neural precursor cells there are in the developing brain is unknown. However, several distinct cell types may exist:
a unipotential precursor to neurons only (“committed neuronal progenitor” or “neuroblast”),
a unipotential precursor to oligodendrocytes only (“oligodendroblast”),
a unipotential precursor to astrocytes only (“astroblast”),
a bipotential precursor that can become either neurons or oligodendrocytes, neurons or astrocytes, and oligodendrocytes or astrocytes, and
a multipotential precursor that maintains the capacity to differentiate into any one of the three cell types.
CNS stem cells are multipotential precursor cells with the innate property to differentiate into all major cell types of the mammalian central nervous system (CNS) including neurons, astrocytes, and oligodendrocytes. The methods for isolation and differentiation of CNS stem cells and the characterization of differentiated cell types have been previously described in detail, U.S. Pat. No. 5,753,506 (Johe). Briefly, CNS stem cells are expanded in serum-free, chemically defined medium containing basic fibroblast growth factor, bFGF, as the sole mitogen. The culture condition permits nearly pure populations of CNS stem cells for a long period both as a mass culture and as a clonal culture.
The mitotic capacity of CNS stem cells, however, is finite. With the previous culture conditions, it had been difficult to expand CNS stem cells beyond about 30 cell-doublings at which point a majority of the cells have lost their capacity for neuronal differentiation and further expand as glial progenitors rather than as multipotential stem cells. The mechanism for this limitation is yet unknown.
We hypothesized that mitotic CNS stem cells secrete an autocrine factor or factors which suppress the entry into cell cycle at the G1 phase of mitosis. This would effectively antagonize the mitogenic actions of bFGF and initiate the differentiation path. Thus, it is a mechanism to self-regulate the proliferation of CNS stem cells and, in vivo, to limit the generation of neurons and glia during development. Consistent with this mechanism is the observation that high cell density promptly differentiates CNS stem cells even in the presence of bFGF and regardless of the passage time.
Although the 30 cell-doublings yield 109-fold expansion of cells, a method for further significant expansion of CNS stem cells would be of significant commercial value. Here, we disclose that constitutive activation of c-myc protein in CNS stem cells prevents their spontaneous differentiation at high cell density, confers resistance to glial differentiation, and increases the mitotic capacity over 60 cell-doublings. This procedure thus yields more than a 1018-fold expansion of CNS stem cells.